Biomining is, in general terms, the use of microorganisms for the recovery of metals from minerals. Its most traditional expression is bioleaching, but we understand biomining as encompassing not only this process, but also the monitoring and intervention of the involved microorganisms, as these techniques are complex and subjected to permanent development; laboratory level research associated to the improvement of processes or the development of new methodologies are also included.
Bioleaching is defined as a method to solubilize metals from complex matrixes in an acid medium using direct or indirect microorganism action. Microorganisms that are useful in these processes belong both to Bacteria and Archaea domains and fulfill two basic conditions: they are acidophiles and chemolithotrophic.
Microorganisms Associated with Bioleaching Processes.
Many microorganisms have been described as being useful in bioleaching processes, among which we can identify genera Acidiphilium spp., Leptospirillum spp., Sulfobacillus spp., Acidithiobacillus spp. and species Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans belonging to Bacteria domain. From Archaea domain we can identify genera Acidianus spp., Ferroplasma spp., Metallosphaera spp., Sulfolobus spp. and Thermoplasma spp. (Rawlings D E. Annu Rev Microbiol. 2002; 56:65-91; Rawlings D E. Microb Cell Fact. 2005; 4(1):13).
Factors Determining Diversity and Metabolic Activity of the Microbiological Community Associated to Bioleaching Processes.
The microorganisms belonging to each above mentioned genus and species produce compounds that increase the rate of different chemical reactions, which allows carrying out bioleaching processes in much shorter times. For this, microorganisms require in their turn a suitable environment to promote said reactions that, for instance, could be aerobic or anaerobic, or require some specific nutrient. Therefore, the environmental conditions under which the bioleaching process is carried out modify the activity and microbiological composition of the present community.
It has been proposed that microorganism participation in bioleaching processes could be direct or indirect (Rawlings D E. Microb Cell Fact. 2005; 4(1):13). It is direct when microorganisms act directly over the metal or over its counter-ion, in either case releasing one ion of the desired metal. On the other hand, the participation is indirect when the microorganism does not use the desired metal or its counter-ion as a substrate, but generates chemical conditions that accelerate or favor said metal solubilization, either by acidification of the medium (e.g., by generating sulfuric acid) or by generating an oxidizing agent that finally interacts with the salt (metal and counter-ion) to be solubilized. For instance, species belonging to genus Acidithiobacillus are able to produce elements that increase the oxidation rate of reduced sulfur compounds (such as sulfide, elemental sulfur, thionates, etc.) by using oxygen as electron acceptor. During this process they generate sulfuric acid as final product and reducing species such as sulfite and thiosulfate as intermediate products, which allows solubilizing sulfur associated metals in the mineral. In particular, Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans contribute with biological components that favor iron (II) to iron (III) oxidation using oxygen as electron acceptor. Generated iron (III) is a major oxidizing agent that can oxidize present sulfides or any other compound to be oxidized.
The common mining practice in bioleaching processes is to leave a heap of mineral in an acid medium, generally sulfuric acid, and gradually remove the acid medium to recover the metal by electrolysis. Often efficient metal recovery yield heaps and “inefficient” heaps are obtained, these latter having a lower yield under the same operation conditions and characteristics of the leached substrate. Being bioleaching a microbiological process, differences of efficiency levels between heaps could be consequence of differences in abundance and type of species in the microbiological community composing them. In this way, this low yield problem could be solved, for example, by inoculating microorganisms that produce components favoring the desired reaction to be maintained during the process. Nevertheless, up to date there is no method allowing the fast, specific and simultaneous identification of bioleaching microorganisms present in a sample.
Microorganisms Detection Method.
According to the state of the art, if it would be desired to determine the major components of a microbiological population with biomining relevance that are present in a sample, it could be done using techniques such as: denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), or selective cultures. These techniques are labor intensive, expensive and require highly qualified workers to perform them. For example, DGGE assay is slow, its completion taking about 3 weeks, and have low sensitivity (detection limit: 104 microorganisms/ml), which is inadequate for biomining communities, as normally there are relevant taxons at lower concentrations than said detection limit. PCR technique also has problems, requiring firstly an individual reaction for each of the species to be determined, which is slow and extremely laborious. In second place, when working with a metagenomic sample, there is the risk of primers having cross-reactivity with other of the species found in the sample, giving both false positive and false negative results. Finally, analysis of microbiological communities using the traditional identification method by culturing has the risk of some species that are present in lower proportion being lost in the process and not being detected. This risk is always present in the case of selective cultures, but is increased when dealing with biomining microorganisms, as the conditions that maintain the growth of these microorganisms are hard to achieve, and some of them are definitely not even able to be cultured in the laboratory. Other problem of the analysis by culture is the slow development of the process, which could take many days.
In consequence, in the state of the art there is no simultaneous detection method for many organisms that is simultaneously fast, specific and cheap.
By mean of the present invention said technical problem has been solved by creating a simultaneous identification method for biomining microorganisms using a DNA fragment array technique.
A good definition of DNA array is that proposed by Schena et al. (Trends Biotechnol. 16, 301-306): “a microscopic ordered nucleic acid array that allows simultaneous analysis of complex DNA samples” (Schena M., Heller, R. A., Theriault, P., Konrad, K., Lachenmeier, E. and Davis, R. W. (1998)). Depending on the diameter of the deposited DNA spots, there are 2 array types: macro-arrays (300 microns or more) and micro-arrays (less than 100 microns). The first can be manually manufactured in the laboratory and the spots can be observed without the help of special equipment. The second require an automated deposition process (normally a robotic deposition platform) and a specialized image acquisition and processing equipment.
In this particular case, DNA fragment arrays comprise an ordered series of spots deposited on a flat surface, such as a glass, silicon or nylon sheet, where every spot contains a large amount of copies of a known DNA fragment that is specific for a determined microorganism with biomining relevance.
The selection method using DNA fragment arrays comprise a simultaneous hybridization of the set of array “spots” with a labeled DNA extract of the studied sample. Normally, DNA from the sample, which has been labeled and fragmented as required, is subjected to a denaturation stage wherein the double stranded DNA is separated, e.g. by heating. When temperature is lowered, DNA will tend to hybridizes with its most complementary fragment according to its physicochemical characteristics. Being this DNA in contact with the array, if there is coincidence between sample DNA and the DNA fragment contained in a spot, labeled sample DNA copies will specifically attach to said spot with the largest possibility. This is due to the larger amount of complementary DNA copies contained in the array spot. In the acquisition and processing stage of the hybridized array image, this label will allow the detection of the microorganisms present in the studied sample.
DNA labeling can be done by any known labeling technique, being fluorescence and radioactive labeling the most common ones.
Arrays and their usage method are known, and we find examples of arrays in the state of the art used to detect the presence of microorganisms in a sample, but none of them is directed to microorganisms that are relevant in biomining.
At the present time, diverse published protocols exist for the manufacture of DNA fragment arrays, and there are also laboratories that offer manufacturing services for this type of arrays. Consequently, only the selection of genes and the design of used DNA fragments defines the specificity and utility of an array, as the manufacture can vary according to the matrix, the method used to bind DNA fragments to the matrix, the spatial distribution of the spots on the matrix, etc., depending on the manufacturing company or the protocol used to manufacture the array in the laboratory (Ye et al. Journal of Microbiological Methods 47 (2001): 257-272).